The detection, identification and quantitation of paraproteins is useful for the detection of multiple myeloma. Monitoring paraprotein production is a necessary aspect to treat such diseases. Those suffering from multiple myeloma will produce one or more abnormal immunoglobulins or paraproteins which, if detected at an early stage, allows an aggressive treatment plan to be employed. Left undetected, a more extreme therapy can be required. Thus, it is important to properly detect paraproteins at as low a level as possible.
Detection of paraproteins may be performed using gel electrophoresis or capillary electrophoresis. In gel electrophoresis, the stained gel generally contains a pattern consisting of a series of dark bands on a light background. This gel response is then visually examined for abnormalities. A gel densitometric trace may also be obtained and used. The gel densitometric trace records the absorbance of the gel at a series of x positions on the gel, using a particular detection wavelength. This trace is useful for quantitation of results.
In capillary electrophoresis, the sample response usually consists of the absorbance of the proteins as they flow past the detector. Capillary electrophoresis has the advantage that separations can be performed in relatively short periods of time by using high voltages, since the small diameter and thin wall of the capillary provide efficient removal of the joule heat generated by the voltage. The capillary electrophoresis measurement is also more readily automated. Typically UV absorbance detection is used for detection of the proteins, with no staining step required.
A response with an x-axis of migration time is typically the data obtained from the capillary electrophoresis experiment. An x-axis that has been shown to give more precise identification and quantitation of electropherogram components is normalized mobility (see, co-pending U.S. application Ser. No. 08/866,282, Likuski, R. K., "Mobility and Normalized Capillary Electrophoresis," incorporated herein by reference). The migration time axis can be changed to normalized mobility by: 1) taking the reciprocal of migration time; 2) multiplying by an appropriate constant; 3) zero correcting by subtracting the electroosmotic velocity; 4) dividing by the zero corrected mobility of a charged marker; and 5) multiplying by a constant, preferably -1. The appearance of this normalized data set also more closely resembled that of the analogous gel electrophoresis densitometer trace, a shape that is familiar to clinicians.
It is common practice to identify or type a paraprotein by its heavy chain and light chain constituent parts. A typical antibody or immunoglobulin consists of a pair of two "heavy" chains linked to a pair of two identical "light" chains to form a hypothetical "Y" structure. The heavy chains form the base of the "Y," and the light chains form the two branches. The heavy chains and light chains are separately synthesized by the immune system. There are two types of light chains, referred to as "kappa" (".kappa.") and "lambda" (".lambda."). Similarly, there are several classes of heavy chains: .gamma. ("IgG"); .alpha. "IgA"); .delta. ("IgD"); .mu. ("IgM") and .epsilon. ("IgE"). IgG, IgA and IgM are the major serum immunoglobulins; IgD and IgE are generally present in serum only at very low concentrations.
Immunofixation electrophoresis (IFE) has been the method of choice for the typing of paraproteins in gels. In IFE, several replicates of sample are subjected to electrophoresis. After gel separation of the components of interest, a different specific antibody is added to each sample replicate. The sample replicates are then allowed to bind to the different specific antibodies. The antibody-protein complex that forms is an insoluble precipitate. Unbound antibody and unreacted protein is then washed away, and the gel is stained, leaving a series of dark bands indicative of the identity of the components present in the original sample. IFE is a reliable, but time consuming and labor-intensive process that is more amenable to gel electrophoresis than capillary electrophoresis.
A related accepted method for the identification of paraproteins is the method of Aguzzi and Poggi (see, Aguzzi et al., "Immunosubtraction Electrophoresis: A Single Method for Identifying Specific Proteins Producing the Cellulose Acetate Electropherogram," Estratto dal. Boll 1.sup.st Sieroter, Milanese 56/3:212-216 (1977) incorporated herein by reference). This method uses cellulose acetate sheets and/or strips. Some strips are left untreated, while others are constructed to contain a segment containing antibodies to sample components of interest near the point of sample application. Using their electrophoretic conditions, the antibodies contained in the constructed segment do not migrate significantly. Serum samples are applied to treated and untreated strips and electrophoresed. In the treated strips, the component of interest present in the sample binds to the appropriate antibody contained in the constructed segment, and the bound antibody-antigen complex precipitates. The component of interest does not migrate past this zone in the treated strips, while migrating normally in the untreated strips. The unbound sample components migrate normally on both treated and untreated strips, and appear in their expected locations. By comparison of the migration patterns of treated and untreated strips, the location of the component of interest can be found. The authors refer to this procedure as immunosubtraction.
The method of Aguzzi and Poggi has been used for the identification of paraproteins in capillary electrophoresis. A sample is first run, and paraprotein(s) visually detected. Antibodies to the components of interest (IgG, IgM, IgA, kappa and lambda) are coated onto beads or left free in solution (see, U.S. Pat. Nos. 5,228,960 and 5,567,282, incorporated herein by reference). The antibodies to the paraproteins, in essence anti-antibodies, are successively added to aliquots of the sample, one type of antibody per sample, causing removal of these components from the sample aliquot, or causing a shift in mobility of the component(s) of interest. These sample aliquots are then run by capillary electrophoresis, and the differences between the untreated and treated samples examined visually to determine the type of paraprotein originally present.
Prior to the discoveries underlying the present invention, methods for identification of paraproteins by immunosubtraction have relied on visual comparison of the differences between untreated and treated samples. Methods relying on visual comparison of results are inherently subjective, and require a time-consuming examination of each sample result. A large paraprotein response can be readily detected and identified visually, but smaller paraproteins can be more of a challenge. The reliability of the method varies with the expertise of the technician examining the immunosubtraction results. What is needed in the art is a process that performs this comparison in an automated fashion and provides a method that is more amenable to high throughput screening, and gives more consistent results. The present invention fulfills this and other needs.